Since the advent of recombinant DNA, methods have been developed and improved for the purification of DNA and RNA to further molecular biology research. While these methods have allowed considerable study of nucleic acids in research environments, they have not addressed issues involved in the human clinical use of purified nucleic acids such as is required for many current gene therapy protocols.
Gene therapy involves the introduction of nucleic acid into a patient's cells, which, when expressed, provide a therapeutic benefit to the patient. Examples include the introduction of an exogenous, functional gene to correct a genetic defect in a patient carrying a defective gene or to compensate for a gene that is not expressed at sufficient levels. Other examples include the introduction of mutant genes, antisense sequences or ribozymes to block a genetic function, e.g., in the treatment of viral infections or cancer.
Much of the focus in gene therapy has been on using viral vectors, especially retroviral vectors, for introducing exogenous nucleic acid into a patient's cells. To date, most of these protocols have been for ex vivo gene therapy, in which the patient's cells are first removed from the patient, genetically modified ex vivo, and then returned to the patient. The alternative to ex viva gene therapy is in vivo gene therapy. In vivo gene therapy refers to the introduction of exogenous genetic capability directly to the patient where it is taken up by the target cells, which then express the introduced gene to produce a therapeutic product. Viral vectors have been used for in vivo gene therapy although their use is associated with a number of drawbacks, e.g. immunogenicity of the viral vector and safety concerns such as insertional mutagenesis or viral contamination.
Other means of in vivo gene delivery include the introduction of naked DNA into the target issue of interest, or the use of lipid-mediated DNA delivery. Typically, introduction of naked DNA will be used when the exogenous genetic capability is to be introduced directly into the target issue. By complexing with liposomes or lipids, DNA is compacted, allowing systemic delivery of the lipid/DNA complexes to various tissues of interest. See PCT patent application WO 93/25673. Lipid/DNA complexes can be targeted to particular tissues by altering the lipid composition, lipid/DNA ratio, mode of delivery, etc.
For any application in which nucleic acid is introduced into a patient, there is a need to produce highly purified, pharmaceutical grade nucleic acid. Such purified nucleic acid must meet drug quality standards of safety, potency and efficacy. In addition, it is desirable to have a scaleable process that can be used to produce large quantities of DNA, e.g., in the range of 100s of milligrams to 100s of grams. Thus, it is desirable to have a process for producing highly pure nucleic acid that does not use toxic chemicals, known mutagens, organic solvents, or other reagents that would compromise the safety or efficacy of the resulting nucleic acid, or make scale-up difficult or impractical. It is also desirable to prepare nucleic acids free from contaminating endotoxins, which if administered to a patient could elicit a toxic response. Removal of contaminating endotoxins is particularly important where the nucleic acid is purified from gram negative bacterial sources, e.g. plasmid or bacteriophage DNA, which have high levels of endotoxins.
The invention described below meets these needs and provides other related advantages as well.